In the various processes of culturing or fermenting microorganisms, it is sometimes necessary at the conclusion of the growth of the culture or the conclusion of the fermentation process to be able to kill the active cells in the mixture so that the growth activity is stopped and the desired product can be recovered from the culture or fermentation mixture. This is particularly true when organisms containing recombinant DNA are grown as production hosts and it is necessary to prevent any viable recombinant organisms from being released into the environment.
It is sometimes desirable to also lyse the cells at the time the cells are killed in order to recover any desired product which is produced intracellularly. One conventional way that cells are killed and lysed is by the use of heat. U.S. Pat. No. 4,601,986 to Wegner, et al. is an example of the use of heat to kill the cells and stop the growth of microorganism cultures. Another method useful on certain microorganisms is to change the osmotic pressure which causes the cells to lyse. An example of this method is illustrated in U.S. Pat. No. 4,299,858 to Aubert, et al. Another conventional method used for lysing cells is by introduction of enzymes which break down the cell walls or membranes. Examples of this method are disclosed in U.S. Pat. No. 3,816,260 to Sugiyama, U.S. Pat. No. 3,890,198 to Kobayashi, et al. and U.S. Pat. No. 3,917,510 to Kitamura, et al. The disclosures of the above patents are incorporated herein by reference.
However, in other instances it is desirable to simply kill the cells to stop the microorganism activity without lysing the cells. This is particularly true in systems where the cells manufacture and secrete the desired product. In such systems it is very desirable to kill the cells without lysing the cells, because lysing the cells releases additional cell debris and materials, thus making recovery and purification of the desired secreted product more difficult and costly. Therefore, when the cells in such a system can be killed without lysing them, process efficiencies in recovery and purification of the secreted products are recognized.
In many systems the host microorganism is difficult to kill, for example fungi. Conventional methods, such as heat, are too severe and will destroy or alter the desired secreted product before the cells are killed. In such systems the product must be recovered without killing the cells, which requires the use of tedious and costly containment procedures and equipment.
In large scale commercial fermentation processes, it is desirable to have more efficient and faster methods for killing the cells and stopping the cell growth so the resulting fermentation mixture can be processed to extract and recover the desired product being produced without lysing the cells thus eliminating the need for containment. The heat method and other known methods for killing cells are too slow and energy inefficient for commercial use and often result in unwanted lysing of the cells. In addition, many of the conventional methods for killing cells are not compatible with culture and fermentation processes for microbial production of enzymes. Conventional methods frequently denature or alter the desired enzyme before it can be isolated and recovered, or those methods introduce materials, e.g., other enzymes, which make the isolation, recovery and purification of the desired enzyme product more difficult, less efficient and, consequently, more expensive.
It is, therefore, an object of this invention to provide a faster, more efficient method for killing cells. It is a further object of this invention to facilitate the extraction and product recovery processing of enzyme products found in a fermentation or culture mixture or medium. It is still a further object of this invention to provide a method for effecting desired cell kill which is compatible with microbial production of enzymes and the recovery and purification of such microbially produced enzymes.